Summary: Despite extraordinary medical advances in the last century, infectious diseases remain the second leading cause of death worldwide, and disease-associated morbidity causes immeasurable harm. The development of new vaccines is, therefore, an imperative for improving global health. The ability of adaptive immune cells to respond more rigorously after a primary exposure, preventing pathology, and in essence ?remembering? the infection is the basis for vaccination. A heterogeneous population of long-lived memory T cells mediate protection from reinfection with previously encountered pathogens; keep chronic, opportunistic and latent pathogens at bay; and defend against tumor growth and metastases. Thus, understanding how memory lymphocytes are induced and sustained is of central importance in the rational design of new vaccines. Tissue- resident memory T cells provide sentinel protection at body surfaces such as the intestinal epithelium, and provide a first line of adaptive immune defense to reinfection. While we now know that tissue-resident T memory cells provide an essential component of immune memory, the transcriptional pathways regulating their formation, survival, and function, and how these relate to those that promote the formation of other memory populations are poorly understood. Improving our understanding of these topics will allow us to harness the immediate protective capacity of this vital memory T cell population in tissues where infections typically begin and modulate activity in the context of immunopathology. To this end, we present a preliminary in vivo functional screen that identifies numerous new regulators of CD8+ tissue-resident memory T cell differentiation. Based on these novel findings, we propose to: (1) Study how Runx3 and Blimp1 support the formation, homeostasis, and function of the CD8+ tissue-resident memory T cell population. (2) Delineate the role of the bromodomain and extraterminal domain (BET) epigenetic ?reader? of histone acetylation, Brd4, in regulating gene expression during memory T cell differentiation. (3) Define the transcriptional network that regulates CD8+ tissue-resident memory T cell formation. By using single-cell sequencing and comparison of open chromatin regions in CD8+ tissue-resident memory T cell populations to predict important transcriptional regulators, we will expand our in vivo screening strategy to identify regulators of CD8+ tissue-resident memory cells. We will leverage the results to enhance tissue-resident memory activity in the context of tumor growth and viral infection. By discovering the transcriptional program and molecular regulators promoting differentiation, survival, and function of tissue- resident memory T cells, we will identify novel targets that can be exploited in the strategic design of therapeutic and protective vaccines, the development of which are of crucial importance to human health.